Reconstruction and analysis of central metabolism for the thermophilic bacterium Thermus thermophilus

Department: University of Delaware, Department of Chemical Engineering

Publisher: University of Delaware

Date Issued: 2010

Abstract: Thermus thermophilus is a thermophilic bacterium that thrives naturally at temperatures around 70-80̊C. It has immense potential in the field of biotechnology because of its unique physical and biochemical properties, especially for production of low-boiling biofuels at low cost. A major limitation for the application of this organism in biotechnology is the limited knowledge regarding its metabolism. Hence, the focus of this project was to elucidate the active metabolic pathways to allow future engineering of this bacterium for biofuel applications. In this study, we have reconstructed and validated the metabolic network model for T. thermophilus HB8 using genome-scale modeling approaches and 13C tracer experiments. First, T. thermophilus HB8 was successfully grown in custom designed miniature bioreactors (~5-10 mL) on defined medium at 70̊C. The maximum growth rate was 0.093 hr-1 with glucose as the sole carbon and energy source. Next, a metabolic network model for T. thermophilus HB8 was constructed using the annotated genome from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The model was validated through stable 13C-isotope tracer experiments. Eight 13C labeled glucose tracers i.e. [1-13C], [2-13C], [3-13C], [4-13C], [5-13C], [6-13C], [1, 2-13C] and [U-13C] glucose were employed for metabolic flux analysis. For each tracer experiment, the enrichments of 29 amino acid fragments were measured using gas chromatography-mass spectrometry (GC-MS). In addition, enrichment of CO2 in the off-gas was measured by on-line mass spectrometer. The isotopomer data were entered into a computational software, Metran, for flux analysis. Fluxes were obtained by fitting the isotopomer data to the reconstructed metabolic model. For the best fits, 95% confidence intervals for all fluxes were computed and compared across all the tracer experiments to determine the pathways that were active in the cells. The comprehensive model for T. thermophilus based on the genome-scale model reconstruction and the concurrent results obtained from tracer studies included 75 reactions and 73 metabolites. The pathways found to be present were: glycolysis, complete citric acid cycle, non-oxidative pentose phosphate pathway, glyoxylate shunt and a complete set of amino acid biosynthesis pathways. The results obtained in this work will allow future metabolic engineering of T. thermophilus and may also be useful for the analysis of other extremophiles.